1. Field of the Invention
This invention relates to a process for readily isolating and recovering highly pure erythritol at a high crystallization yield from a culture medium of an erythritol-producing bacterium (microorganism), which comprises readily separating and removing various impurities and by-products, for example, various salts, coloring materials and polysaccharides originating from starting raw materials and from additives added to the medium.
2. Prior Art
The term "erythritol" used herein exactly means meso-erythritol which is useful as a sweetener or as an intermediate in the preparation of various drugs or industrial chemicals.
Examples of known erythritol-producing bacteria which produce erythritol through fermentation include those belonging to the genus Aureobasidium producing glycerol as the main by-product (JP-A-61-31091, the term "JP-A" herein used mean unexamined and published Japanese Patent application); Moniliella tomentosa var. pollinis producing glycerol and ribitol which is a sugar alcohol carrying five carbon atoms (JP-A-60-110295.about.110298), Candida Zeylonoides (ATCC 15585) and Torulopsis famata (ATCC 15586) (JP-A-49-118889); Candida lypolytica (U.S.P. 3,756,917); and those belonging to the genera Trigonopsis and Candida (JP-B-47-41549, the term "JP-B" herein used means examined Japanese patent publication.
A conventional process for isolating and recovering erythritol from a culture medium obtained by culturing one of these erythritol-producing bacteria in an aqueous medium comprises subjecting said culture medium to a pretreatment such as decoloration with the use of activated carbon, desalting and decolorizing the culture medium with ion exchange resins and then concentrating and cooling the same to thereby crystallize the aimed erythritol.
During the culture of an erythritol-producing bacterium, a large amount of inorganic salts such as KH.sub.2 PO.sub.4, MgSO.sub.4, CaCl.sub.2, K.sub.2 SO.sub.4, CaSO.sub.4, FeSO.sub.4, MnSO.sub.4, ZnSO.sub.4 or (NH.sub.4).sub.2 HPO.sub.4 and nitrogen sources such as (NH.sub.4).sub.2 SO.sub.4, urea, NH.sub.4 Cl or NH.sub.4 NO.sub.3 are added to the medium. Further a large amount of nutritional sources such as corn steep liquor, soybean meal, various amino acids, peptone, thiamin or yeast extract are added thereto. When corn steep liquor is added, a particularly remarkable coloration of the culture medium is observed. Therefore the conventional process as described above is disadvantageous, because a large amount of activated carbon and/or ion exchange resins as well as chemicals for regenerating the same are required in order to completely decolorize or desalt the impurities originating from these additives.
In addition, various by-products such as glycerol, ribitol or polysaccharides are formed during the course of the culture in the abovementioned process. When crystalline sucrose or crystalline glucose to be used as a starting raw material is substituted with refined glucose obtained by, for example, enzymatic saccharification of starch, which comprises 93 to 97% of glucose and the residual amount of oligosaccharides such as disaccharides, trisaccharides or higher ones, the oligosaccharides contained in the starting raw material would remain as impurities in the culture medium. These impurities can not be removed by treating the culture medium with activated carbon or decolorizing or desalting the same with the use of ion exchange resins. When the culture medium is concentrated to thereby improve the crystallization yield of the erythritol, the concentration of these impurities is also increased. As a result, the concentrate to be crystallized becomes highly viscous, just like corn syrup, which considerably lowers the crystallization rate of the erythritol. Therefore it is unavoidable to concentrate the culture medium to a limited extent, which significantly lowers the crystallization yield of the aimed erythritol.
Under these circumstances, we have found that the impurities affecting the crystallization of erythritol mainly comprise the following constituents.
(i) Glycerol formed by a side reaction. PA1 (ii) By-products other than glycerol. PA1 (iii) When refined glucose obtained by, for example, enzymatic saccharification of starch is used as a starting raw material, oligosaccharides including disaccharides and higher ones contained in the starting glucose as well as reaction products formed therefrom. PA1 (iv) Polysaccharides comprising glucose as the main constituent and having .beta.-1,4 bonds.
When the culture medium containing a large amount of the impurities (i), (ii) and (iii) is decolorized/desalted with the use of activated carbon and ion exchange resins in a conventional manner and then crystallized by concentrating, the mother liquor would show a rapid increase in viscosity and thus becomes just like corn syrup before a satisfactory crystallization yield is achieved. As a result, the crystallization rate is considerably lowered and it becomes difficult to separate the precipitated crystals from the mother liquor.
The polysaccharides, i.e., the impurities (iv) have high molecular weights of several thousands to several ten thousands and thus formed at a low concentration, i.e., 30 to 500 ppm. However it is impossible to remove them by conventional methods such as decolorization/desalting with the use of activated carbon or ion exchange resins. Therefore these polysaccharides would be precipitated during the crystallization of erythritol. As a result, the obtained erythritol crystals are contaminated with the polysaccharides. Thus an aqueous solution of the obtained erythritol crystals is turbid, which lowers the qualities.
In order to improve the crystallization yield of erythritol, it is possible to substitute the starting glucose obtained by enzymatic saccharification of starch which contains a large amount of oligosaccharides including disaccharides and higher ones with crystalline glucose or crystalline sucrose to thereby lower the content of the oligosaccharides originating from the starting raw material as well as products formed therefrom, namely the impurities (iii) as defined above. However this method is also unsatisfactory, since the culture medium still contains the impurities (ii), i.e., by-products other than glycerol.
Further there have been proposed processes for separating ethylene glycol from a solution containing the same together with coloring materials and organic acids which comprise subjecting to said solution to chromatography with the use of a cation exchange resin of, for example, a sodium salt type as an separation medium (JP-A-57-106632, 57-142930 and 57-142931). The application of each of these processes; wherein water is used as an eluent, is limited to the separation of ethylene glycol from organic acid salts and coloring materials.
Furthermore there have been shown elusion curves formed by ion exclusion of sodium chloride/glycerol (Gupta, 1971) and sodium chloride/glucose (Singh, 1978) with the use of sodium type cation exchange resin as a separation medium (Prem C. Nlgam et al., Studies on Ion-Exclusion Phenomena, Ind. Eng. Chem. Process Des. Dev. 20, 182-188 (1981); Gupta A. K. M, Tech. Thesis, IIT, Kanpur, India (1977); and Singh, D.M., Tech. Thesis, IIT, Kanpur, India (1978).
However these references neither describe nor suggest the process of the present invention which comprises subjecting an erythritol-containing culture medium to chromatography with the use of an alkali metal or ammonium type cation exchange resin as an separation medium to thereby efficiently remove various salts, coloring materials, various oligosaccharides and polysaccharides from said culture medium and thus isolating and recovering highly pure erythritol crystals at a high crystallization yield.
We have further conducted extensive studies to overcome the above problems and consequently succeeded in achieving the object by carrying out chromatography with the use of alkali metal or ammonium type strongly acidic cation exchange resins as separating mediums.
The separation capability of the strongly acidic cation exchange resins to be used in the present invention would be lowered when used repeatedly. However it can be readily restored by washing the cation exchange resins with warm alkali solutions, which enable the prolonged continuous operation of the process of the present invention.